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Journal of Pharmaceutical and Biomedical Analysis 92 (2014) 90–97

Contents lists available at ScienceDirect

Journal of Pharmaceutical and Biomedical Analysis

j o ur nal ho me page: www.elsev ier .com/ lo cate / jpba

A dynamic thermal ATR-FTIR/chemometric approach to the analysisof polymorphic interconversions. Cimetidine as a model drug

Natalia L. Calvo, Rubén M. Maggio, Teodoro S. Kaufman ∗

Area Análisis de Medicamentos, Departamento Química Orgánica, Facultad de Ciencias Bioquímicas y Farmacéuticas, Universidad Nacional de Rosario andInstituto de Química Rosario (IQUIR, CONICET-UNR), Suipacha 531, Rosario S2002LRK, Argentina

a r t i c l e i n f o

Article history:Received 18 October 2013Received in revised form21 December 2013Accepted 27 December 2013Available online 7 January 2014

Keywords:Crystal polymorphismMultivariate curve resolution withalternating least squares (MCR-ALS)CimetidineATR-FTIR/chemometricsCrystal form interconversion

a b s t r a c t

Crystal polymorphism of active ingredients is relevant to the pharmaceutical industry, since polymorphicchanges taking place during manufacture or storage of pharmaceutical formulations can affect criticalproperties of the products. Cimetidine (CIM) has several relevant solid state forms, including four poly-morphs (A, B, C and D), an amorphous form (AM) and a monohydrate (M1). Dehydration of M1 has beenreported to yield mixtures of polymorphs A, B and C or just a single form.

Standards of the solid forms of CIM were prepared and unequivocally characterized by FTIR spec-troscopy, digital microscopy, differential scanning calorimetry and solid state 13C NMR spectroscopy.Multivariate curve resolution with alternating least squares (MCR-ALS) was coupled to variable tem-perature attenuated total reflectance Fourier transform infrared spectroscopy (ATR-FTIR) to dynamicallycharacterize the behavior of form M1 of CIM over a temperature range from ambient to 160 ◦C, withoutsample pretreatment.

MCR-ALS analysis of ATR-FTIR spectra obtained from the tested solid under variable temperature con-ditions unveiled the pure spectra of the species involved in the polymorphic transitions. This allowedthe simultaneous observation of thermochemical and thermophysical events associated to the changesinvolved in the solid forms, enabling their unequivocal identification and improving the understandingof their thermal behavior.

It was demonstrated that under the experimental conditions, dehydration of M1 initially results in theformation of polymorph B; after melting and upon cooling, the latter yields an amorphous solid (AM).It was concluded that the ATR-FTIR/MCR association is a promising and useful technique for monitoringsolid-state phase transformations.

© 2014 Elsevier B.V. All rights reserved.

1. Introduction

The quality of pharmaceutical products is currently assured onthe basis of a scientific understanding of their physical and chemicalproperties that represent quality attributes. Crystal polymorphismis a property of the solid state by which a compound exhibits dif-ferent solid crystalline phases, resulting from at least two differentmolecular arrangements. In Pharmaceutics, the term polymor-phism is often employed to cover a variety of solids, includingcrystalline, amorphous, and solvate/hydrate forms. In that sense,amorphous forms are special cases of polymorphs, characterizedby the absence of a regular crystal structure; additionally, solvatesare often termed pseudo-polymorphs [1].

The polymorphs display different mechanical and physicalproperties, including crystal habit, lattice energy, intermolecular

∗ Corresponding author. Tel.: +54 341 4370477x118; fax: +54 341 4370477x112.E-mail address: [email protected] (T.S. Kaufman).

interactions, particle density, thermodynamic activity, meltingpoint, solubility, stability, and dissolution rate [2]. These differencesmay have impact on the production process, changing manufac-turing reproducibility and product performance, affecting drugdissolution, absorption and bioavailability [3].

The rational control of polymorphs of active pharmaceuticalingredients has been an important goal for the pharmaceuticalindustry; hence, proper characterization of the polymorphs isneeded in order to gain better understanding of the physicochem-ical characteristics of the bulk drugs, which may influence theirprocessability and capability to withstand the manufacturing pro-cesses and their stability during the storage period [4–6]. Theinability to fully characterize drug polymorphic forms has led inthe past to a number of problems, including the case of the proteaseinhibitor ritonavir, where formation of a previously unknown poly-morph during the manufacturing stage led to a significant delay inthe supplies of the capsule form [7].

Since polymorphs have different lattice energies, the less stableand more energetic species tend to convert to the most stable, less

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N.L. Calvo et al. / Journal of Pharmaceutical and Biomedical Analysis 92 (2014) 90–97 91

Fig. 1. (A) Chemical structure of Cimetidine. Optical microscopy of the polymorphs of cimetidine. (B) Form A. (C) Form B. (D) Form C. (E) Form D. (F) Form M1.

energetic, and likely the less soluble, crystal form. These transfor-mations can take place during manufacturing operations (grinding,kneading and tableting); however, storage variables (temperatureand humidity) may affect the stability of metastable crystal forms,and pharmaceutical excipients can also contribute to polymorphicconversions [8].

Cimetidine (CIM), one of the first blockbuster drugs,is (Z)-N′′-cyano-N-methyl-N′[2-[[(5-methyl-1H-imidazol-4-yl)methyl]thio]ethyl]guanidine (Fig. 1A). The drug is a specificcompetitive antagonist of the histamine H2-receptor at the pari-etal cells [9], which inhibits the histamine-stimulated secretionof gastric acid and reduces pepsin output. Hence, CIM has beenwidely used in conditions where inhibition of gastric acid secretionmay be beneficial, such as heartburn associated with acid reflux,duodenal and gastric ulcers, gastroesophageal reflux disease, andhypersecretory syndromes such as the Zollinger–Ellison’s [10]. Inaddition, the drug is currently being considered for alternativeindications [11].

Cimetidine has been discussed in the literature as a relevantexample of a polymorphic substance, when crystallized undervarious conditions [12]. Four crystal modifications (A, B, C, D)and monohydrate M1 [13] have been described and character-ized or differentiated by various methods, including infrared (IR),near infrared and Raman spectroscopy [14–17], X-ray diffraction[12,18,19], synchrotron X-ray powder diffraction [20], atomic forcemicroscopy [21], solution calorimetry [22] and isotopic labeling-solid state NMR spectroscopy [23,24].

Polymorphic interconversions of CIM have also been recordedwith isothermal calorimetry [25], and scanning thermalmicroscopy coupled to localized thermal analysis allowed todistinguish each of two forms [26], while differential scanningcalorimetry (DSC) proved to have difficulties in identifying thedifferent cimetidine polymorphs owing to the similarity in theirbulk thermal behavior [27]. The thermodynamic stabilities of theanhydrous forms are extremely close to each other, in terms ofheat of fusion, being hardly distinguished experimentally, so theyhave been considered ‘practically isoenergetic’ [27,28].

A number of additional pseudo-polymorphs [14,26,29,30] havebeen described, but their access could not be easily repeated. Inaddition, preparation of a fifth modification was reported, but theprocedure could not be reproduced even by high-throughput crys-tallization experiments [31].

Interestingly, only three polymorphs were detected in a solventscreen including over 100 different conditions [32], while somepublished powder diffractograms still cannot be clearly attributedto specific crystal modifications [19]. The polymorphs differ in theirbioavailability properties, principally due to their differences in dis-solution rates [33–35]; however, in pharmaceutical formulationsonly modifications A and B are used [26]. Forms A, C, D and M1have been resolved by X-ray crystallography [18,36–38]. It has beenshown that even slight changes in crystallization or processing con-ditions (grinding, milling, etc.), sometimes not noticeable, are ableto produce different crystal forms or their mixtures in an unpre-dictable manner [27]. In some cases, the obtained structures areidentical; however their crystal parameters of the unit cell aresomewhat different [16]. The presence of polymorphic mixturesin the commercial CIM product has been reported [38].

On the other side, it has been informed that, upon dehydra-tion, the monohydrate M1 can be transformed into form A [19,24],or into forms A, B and C or a mixture of them, in an irrepro-ducible manner, or to the modification already enclosed as animpurity [27].

Vibrational spectroscopy is a green tool for multicomponentanalysis [39], which has proven to be suitable for measuring powderor polycrystalline samples for which even subtle changes in molec-ular structure need to be detected. Interestingly, however, in somecases FTIR analysis alone was unable to ascertain the compositionof polymorphic forms of CIM [38].

Chemometric strategies are playing an increasingly impor-tant role in the analysis of multi-component mixtures and inthe understanding of the most diverse phenomena. Several mul-tivariate exploratory and resolution methods can be appliedto the analysis of evolving mixtures, to provide informationabout pure compounds and their abundance in a sample; amongthem, the multivariate curve resolution with alternating leastsquares (MCR-ALS) approach is gaining wide acceptance due to itsversatility [40].

The empowering of vibrational spectroscopies by associationwith MCR-ALS for the extraction of valuable information fromcomplex evolving data has demonstrated to be highly successful[41,42]. Therefore, herein we report how a dynamic thermal atten-uated total reflectance-FTIR/chemometrics strategy, with MCR asthe chemometric tool (ATR-FTIR/MCR), may be used for monitor-ing the polymorphic transformation of a solid form, distinguishing


92 N.L. Calvo et al. / Journal of Pharmaceutical and Biomedical Analysis 92 (2014) 90–97

and characterizing the involved polymorphs. This potential isdemonstrated for CIM, especially for the transformation of itsmonohydrate M1.

2. Materials and methods

2.1. Instrumentation

FTIR spectra were acquired in a Shimadzu Prestige 21 spec-trometer (Shimadzu Corp., Kyoto, Japan) over a wavenumber rangeof 4000–600 cm−1. ATR experiments were carried out with adiamond-based ATR accessory (GladiATR, Pike Technologies, Madi-son, USA) fitted with a Pike temperature control unit.

Calorimetric determinations were performed in a Shimadzumodel 60 differential scanning calorimeter (Shimadzu Corp., Kyoto,Japan), operating under a constant flow of nitrogen (30 ml min−1).The sample powders (10 mg) were placed in closed aluminum pansperforated with a pin-hole to equilibrate pressure from potentialexpansion of evolved gases or residual solvents, and heated at arate of 5 ◦C/min between 40 and 160 ◦C. An empty pan was used asa reference.

Melting points were determined with an Electrothermal, model9100 digital melting point instrument (Bibby Scientific Ltd.,Staffordshire, UK). The samples were heated at 10 ◦C/min up to10 ◦C below the temperature of interest; then, the heating ratewas changed to 1 ◦C/min. In the case of M1, the heating rate was1 ◦C/min.

Digital optical microscopy of the crystal forms was carried outwith the aid of a Correct microscope (Seiwa Optical, Tokyo, Japan),fitted with 10×, 30× and 100× objectives and a 5.0 MegapixelsBeion CMOS digital camera [Shanghai Beion Medical TechnologyCo., Ltd., Shangai, China; resolution 2592 × 1944 (H × V)].

The high-resolution cross polarization-magic angle spinning(CP-MAS) 13C solid state NMR spectra for polymorphs B and Cwere recorded using the ramp {1H} → {13C} CP-MAS sequencewith proton decoupling during acquisition. All the solid-state NMRexperiments were performed at room temperature in a BrukerAvance II-300 spectrometer equipped with a 4 mm MAS probe.The operating frequency for protons and carbons was 300.13 and75.46 MHz, respectively. Glycine was used as an external referencefor the 13C spectra and to set the Hartmann–Hahn matching con-dition in the cross-polarization experiments. The recycling timewas 15 s for sample B2 and 20 s for sample C2. Contact time duringCP was 2 ms. The SPINAL64 sequence was used for heteronucleardecoupling during acquisition with a proton field H1H satisfyingω1H/2� = �HH1H = 62 kHz. The spinning rate for all the samples was10 kHz.

2.2. Chemicals

The cimetidine bulk drug employed (polymorph A) was of phar-maceutical grade (BP 2002) and was acquired to Saporiti (BuenosAires, Argentina). During the experiments, the drug was kept in adesiccator, protected from light. Double-distilled water was usedfor the preparation of aqueous solutions. All other chemicals wereof analytical grade.

2.3. Preparation of the polymorphs of cimetidine

The forms A, B, C and D and M1 (monohydrate) were obtainedby crystallization, as follows [14]. Form A was obtained after slowlycooling a hot (60 ◦C) saturated solution of the drug in isopropanol.Form B was obtained after cooling slowly a hot (70 ◦C) 15% aque-ous solution of the drug. Form C was prepared by rapidly coolingto 5 ◦C a 5% aqueous solution of the drug. Form D was obtained byprecipitation from a saturated solution of CIM in methanol/water

(1:1, v/v) adjusted to pH 6 with acetic acid, which after being main-tained at 25 ◦C, was brought to pH 8.5 in an ice bath by additionof concentrated ammonia. The M1 monohydrate was prepared bypouring a hot 15% aqueous solution over three times the amountof ice, prepared from distilled water [12,27,43]. The solids were fil-tered under reduced pressure, and the residue was stripped off ofthe remaining solvent under vacuum. The thus prepared solid formswere stored at room temperature, in dark screw-cap glass bottles.The amorphous form AM was obtained as a stable glassy solid bymelting commercial polymorph A, followed by cooling down theliquid to room temperature [25]. For the experiments, the particlesize of the samples was standardized by passage through a 50 meshsieve.

2.4. FTIR determinations

Polymorph characterization was performed with the samplesprepared as mulls suspended in Fluorolube® or with the pure solids,by the ATR technique. For polymorph conversion experiments, thespectrometer was coupled to the ATR accessory. Data acquisitionwas performed under predefined temperature conditions on 20 mgsamples, at a resolution of 4 cm−1, employing 20 scans per spec-trum. Spectra were saved in .txt format for their further processingand analysis.

2.5. Chemometrics and graphics software

The computing routines involving spectral data manipulationand the MCR-ALS method were run in Matlab R2010a (Mathworks,Natick, USA). Full spectra (4000–600 cm−1) were used withoutfurther processing. Statistical data analyses were performed withOrigin 7.5 (OriginLab Co., Northampton, USA).

3. Theoretical background

The bases of MCR-ALS have been discussed in detail elsewhere[44,45]. A short theoretical background is given here. Boldface cap-ital letters are used for matrices, and lowercase italics are used forscalars. The superscript “T” is employed to denote a transposedmatrix, and ||X|| represents the norm of matrix X.

Assuming that the spectroscopic data fit an additive linearmodel conforming to Beer’s law, the set of m spectra correspondingto mixtures containing contributions from k components acquiredat n different wavenumbers, can be accommodated in a m × nexperimental data matrix (X). In this way, the rows of X representthe spectra acquired in a time sequence, and each element xij in X isthe absorbance of the ith sample, at the jth wavelength. MCR can beused to perform the optimal decomposition of X into the productof two smaller matrices, C and ST, as described in Eq. (1),

X(m×n) = C(m×k)S(n×k)T + E(m×n) (1)

where C is the matrix of the concentration profiles of the pure com-ponents and S is the matrix of their pure spectra. E is the residualmatrix, which contains the information not explained by the model.

The above decomposition is performed employing a strategywhich involves ALS. However, as a consequence of rotational orintensity ambiguity, the resulting solution is not unique, as morethan one set of matrices C and S can reconstruct X by multiplica-tion [46]. Restriction of the number of possible solutions to thoseembodied with physical meaning can be achieved by imposing con-straints.

In addition, MCR-ALS requires initial estimates of C and S, whichwill be used to find a model able to minimize an error criterion, suchas the lack of fit (lof) of the MCR data (x∗

ij) to the experimental values

(xij), as detailed in Eq. (2). Previous knowledge of the system, such



Fig. 2. ATR-FTIR spectra of different forms of CIM in the 1800–600 cm−1 region. Polymorphs A (A), B (B), C (C) and D (D); monohydrate M1 (E) and amorphous AM (F).

as the number of species involved and their corresponding spectra,can be employed to input initial guesses of C and S and add suitableconstraints.

lof (%) = 100 ∗(∑

i

∑j(xij − x∗

ij)2∑

i

∑jx

2ij

)0.5

(2)

Given the initial estimates of C and S, matrix X is decomposedby iteratively solving two alternating least squares problems, theminimization of the error over C for fixed S and the minimizationof error over S for fixed C. The cycle of iterations ends when themodel error reaches a pre-established minimum value, accordingto Eqs. (3) and (4).

Min(C)‖X − CST‖ → C = XS(STS) (3)

Min(ST)‖X − CST‖ → S = XTC(CTC) (4)

Statistical comparison of the MCR-derived spectra for eachspecies with the spectra of the corresponding reference standardsemploying Pearson’s correlation coefficient (r) can be used as anindicator to assess the quality of the results.

4. Results and discussion

4.1. Characterization of the solid forms

Polymorphs A, B, C, D and monohydrate M1 were obtained. Sinceinconsistent nomenclature of the polymorphic forms has been usedthroughout the literature by different authors [14,16,22,31], themodifications were unequivocally characterized by digital opti-cal microscopy, ATR-FTIR spectroscopy, DSC and CP-MAS 13C NMRsolid state spectroscopy. Designation of the polymorphs is accord-ing to Hegedüs and Görög [14].

Digital optical microscopy revealed the crystalline habits of thepolymorphs (Fig. 1). Form A was distinguished as platelets, poly-morph B appeared as fine sharp needles, form C was observed as

short needles, while polymorph D presented prismatic crystals, andM1 was seen as aggregated particles, in full agreement with theliterature, where scanning electron microscopy revealed them toconsist of small pyramidal crystals [19,25].

For ATR-FTIR characterization, the temperature (30 ◦C), particlesize, amount of sample and the pressure exerted on the sample dur-ing the measurement were standardized. Under these conditions(Fig. 2), polymorphs B and C exhibited highly similar spectra. Thissimilarity has led other researchers to suppose that they were thesame substance [15,27]. Taking into account previous reports indi-cating that modification C is hardly reproducible [14] and readilytransformed into form B upon minor mechanical treatment, suchas trituration by hand in an agate mortar [27], in order to ensure itsintegrity extreme precautions regarding these factors were takenwhen polymorph C was handled.

Furthermore, in order to unequivocally ensure their identi-ties, forms B and C were characterized by CP-MAS 13C NMR solidstate spectroscopy (Fig. 3), resulting in full agreement with thosereported by the group of Middleton [23].

In the DSC analysis, the anhydrous forms A, B and D exhib-ited single peaks (Fig. 4A), without shoulders, at 145.0, 147.1 and147.8 ◦C, respectively, corresponding to their melting points.Thesewere in good agreement with data recorded with the melting pointapparatus, where all melted samples solidified to yield a glassyamorphous solid. On the other hand, the monohydrate form M1(Fig. 4B) exhibited an endothermic transition beginning at 58.55 ◦Cwith a peak at 72.30 ◦C, followed by a small exothermal curveat 81.05 ◦C. This was interpreted as a result of the loss of wateraccompanying the transition from M1 to an anhydrous form. Asecond endotherm, at 142.86 ◦C, corresponds to the melting pointof the anhydrous form. Both endothermic transition temperatureswere in agreement with those recorded by the melting point appa-ratus. The calculated heat of transition was 140.12 mJ mg−1, ingood agreement with the literature (140.5 mJ mg−1) [27]. Table 1summarizes the most relevant characteristic data of the differentforms.



Fig. 3. Proton-decoupled CP-MAS 13C NMR spectra (at 75.46 MHz) of polymorphs C (A) and B (B) of CIM.

Fig. 4. DSC behavior of CIM. (A) Forms A, B and D. (B) Monohydrate M1.

4.2. Dynamic thermal ATR-FTIR/chemometrics monitoring of theconversion of M1

The first step in MCR analysis of the spectral data matrix is todetermine its chemical rank, i.e., the number of absorbing speciesor contributing components. Principal component analysis (PCA) isone of the most widely used approaches to estimate the chemicalrank of a series of spectral data.

PCA of the matrix containing the acquired ATR-FTIR data result-ing from monitoring the effect of heating on M1 and inspection ofthe resulting screen plot [47] revealed the presence of three dif-ferent forms (Fig. 5). Therefore, the computation was initializedwith three components, including the spectra of the solid formsM1 and AM. A random vector, which was input to initialize thethird component in order to avoid prediction bias.

MCR constraints, such as non-negativity of spectral concentra-tions and intensities, were also applied in order to confer physicalsense to the results. The closure restriction of concentration values(sum of the abundances of the analytes’ established as 1) was also

Table 1Summary of physical properties of the different forms of CIM.

Form A Form B Form C Form D Monohydrate M1

DSC (◦C)[ET = endotherm]

144.99 147.09 143.44a 147.81 Onset ET1: 58.55ET1: 72.30ET2: 142.86Htrans = 140.12 mJ mg−1

Melting point (◦C) 140.3 142.0 141.9 141.9 69.1 and 141.3

Crystal morphology(optical microscopy)

Thin platelet Fine needle Needle Prism Pyramid

FTIR, characteristicbands (�max, cm−1)

3208 3224 3230 3281 3497

3142 3148 3152 3201 33842923 2941 2943 2906 32982171 2163 2162 2151 21511615 1606 1606 1605 15911579 1582 1581 1579 14491451 1415 1413 1489 14141377 1370 1370 1372 12561201 1227 1227 1306 11631153 1189 1178 1205 10061074 1180 1064 1070 958952 1174 946 952 814665 1064 767 754 634

a Open pan.



Table 2Pearson’s correlation coefficients (r) of the MCR pure spectra and the reference spectra of the solid forms of CIM, in different spectral regions.

Origin of the spectrum Pearson’s r valuesa in the spectral region (cm−1)

MCR spectrum Solid form Full spectrum (4000–600) Fingerprint zone (1650–600) C H/N H stretching (3600–2500)

S1 M1b 0.985 0.976 0.979S2 A 0.814 0.814 0.589

Bb 0.988 0.952 0.953C 0.934 0.897 0.949D 0.844 0.844 0.730

S3 Mb 0.969 0.978 0.991Number of datapoints (n) 3527 1090 1142

a Cut off value for the similarity test at this stage was arbitrarily set at r = 0.950.b Best-matching species.

added, based on the fact that the no change in the number of molesof cimetidine takes place during the studied process.

MCR-ALS performed the spectral deconvolution of the exper-imental data matrix [48] uncovering the temperature-dependentevolution of the three species, and revealing that they coexistedonly in pairs (Fig. 5B).

Statistical comparisons between the spectra of the authenticpure forms of CIM and those offered by MCR-ALS were carried outemploying Pearson’s correlation coefficients (r) between the cor-responding spectral vectors. Correlations were performed in thewhole spectral region (4000–600 cm−1), as well as in the finger-print zone (1650–600 cm−1) and along the 3600–2500 cm−1 range,which contains the fundamental bands corresponding to C H andN H stretching of CIM [49].

As stemmed from the results consigned in Table 2, the spectralinformation recovered after the MCR-ALS deconvolution processwas highly correlated with the spectra of the standards of somesolids. Values exceeding 0.95 were considered satisfactory, ensur-ing selectivity of the time-composition profiles.

Polymorphs B and C exhibited the best correlations. When thefull spectra of both pure compounds were compared with S2, r val-ues of 0.988 and 0.934 were obtained for forms B and C respectively,demonstrating a better overall fit for polymorph B.

Infrared spectra of forms B and C are very similar; however,Hegedüs and Görög [14] found some bands and regions whichshow differential properties and can be employed as diagnostictools to assign polymorphic identity. Therefore, in order to furtherensure the correct assignment of the intermediate form S2 to eitherpolymorph B or C, a similar but more thorough Pearson’s r-basedstrategy was followed, as an additional step.

Fig. 5. Dynamic thermal ATR-FTIR/chemometrics monitoring of the heat-mediatedtransformation of the monohydrate M1 of CIM. (A) MCR-ALS output. Spectra ofthe species involved. (B) Concentration profiles of the different species during theprocess.

In this second instance, informative spectral ranges (Table 3)were examined, including the regions between 3400 and2700 cm−1 (C H/N H stretching), 3009 and 2909 cm−1 (cover-ing the peak at 2959 cm−1 [14]), 1750 and 1650 cm−1 (to includecharacteristic peaks of forms B and C), obtained by differential com-parison between the pure FT-IR spectra), and that between 1243and 1050 cm−1 (to include the characteristic peak at 1180 cm−1

[14]). In all cases, the r-values were higher for form B, demonstrat-ing its better fit to the S2 spectrum provided by MCR. In addition,similar results were observed when the spectra were systematicallydivided into 200 cm−1 bins and compared pairwise.

These results confirmed that the crystal forms involved inthe monitored transformation were the starting monohydrate M1(Fig. 6A), which agreed with S1 (r = 0.985), and a second polymorphformed by dehydration of the monohydrate (Fig. 6B), which wasidentified as form B, taking into account the values of r betweenthe spectrum of form B (r = 0.988) and the spectrum provided byMCR-ALS for S2.

On the other hand, this analysis confirmed that spectrum S3(Fig. 6C) was in good statistical agreement with that of the amor-phous melted form AM (r = 0.969), which began to form whenpolymorph B approached its melting point, and it was observedto solidify upon cooling to give a glassy solid. The lack of fit valuebetween the MCR-ALS resolution results and the original X matrixwas lower than 5%, ensuring the suitability of both, the outputspectra and concentration profiles.

Interestingly, spectra of forms A, B, C and D exhibited character-istic peaks corresponding to the C N stretching band of the nitrilemoiety at 2176 [43], 2171, 2164 and 2150 cm−1, respectively, whileS2 displayed a maximum at 2167 cm−1.

Once the involved species were identified, the transitions amongthe different forms could be easily determined employing theirtime-concentration profiles. The transition temperatures obtainedfor the conversions M1 → B and B → AM were approximately66.0 ◦C and 143.5 ◦C, being in good agreement with the observedDSC plots and with the literature.

The anhydrous forms (A, B, C and D) did not present thermally-induced interconversion into another polymorph before melting;however, all of them melted in the 140–150 ◦C range, furnishing aliquid which solidified to afford a glassy solid (AM), which did notcrystallize.

Table 3Pearson’s r values for the comparison of forms B and C of CIM against S2 along keydifferential spectral regions.

Wavenumberrange (cm−1)

Spectral information Pearson’s r coefficient

Initial Final S2 vs. B S2 vs. C

3400 2701 C H/N H stretching 0.978 0.9233009 2909 Peak at 2959 cm−1 [14] 0.955 0.9111750 1650 Diagnostic region 0.941 0.7601243 1050 Peak at 1180 cm−1 [14] 0.923 0.877



Fig. 6. Stacked graphics for comparison between the spectra of solids forms M1 (A),B (B) and AM (C), and those provided by MCR-ALS deconvolution S1 (A), S2 (B) andS3 (C), respectively.

5. Conclusions

Dynamic thermal ATR-FTIR spectroscopy coupled with MCR-ALS (ATR-FTIR/MCR), provided a valuable alternative for moni-toring the polymorphic transformations of monohydrate M1 ofcimetidine (CIM). Statistical comparison of the ATR-FTIR spectra ofauthentic solid forms of the drug with the spectra provided by MCRwas able to reveal that upon heating, M1 dehydrates to yield poly-morph B at approximately 66.5 ◦C, which agrees with the transitiontemperature previously informed in the literature. In turn, fur-ther increase in the system temperature leads to the conversion ofform B into another species, with a melting point of approximately143.5 ◦C, in accordance with literature data on the melting point ofpolymorph B. The monitoring system also revealed that the melteddrug solidified upon cooling to yield a glassy amorphous solid (AM).

Thus, MCR-ALS analysis of the acquired series of spectra pro-vided an accurate picture of the time evolution of the process, andthis approach was also found to be useful in estimating the underly-ing spectra of the intervening forms, allowing to identify the speciesinvolved at all times, unlike the calorimetric techniques.

The method required only minor amounts of sample, sincecumulative spectral acquisition followed by Fourier transform ofthe data compensated for low signal amplitudes. On the other hand,no sample preparation steps were involved. Therefore, the pro-posed approach should be considered as a useful strategy with highpotential as a tool for gaining insight into the behavior of solid formsof active pharmaceutical ingredients, when they are subjected tothermal stress.

Acknowledgements

The authors are thankful to Secretaría de Ciencia Tecnología eInnovación (SECTeI), Consejo Nacional de Investigaciones Cientí-ficas y Tecnológicas (CONICET), Agencia Nacional de PromociónCientífica y Tecnológica (ANPCyT), Secretaría de Ciencia y Tec-nología de la UNR (SECyT-UNR) for financial support and Dr. JorgeMalarría (IFIR-CONICET) for facilitating access to the DSC equip-ment. NLC acknowledges CONICET for her fellowship.

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